Pleiotrophin (PTN) is a platelet-derived growth factor-inducible heparin-binding growth and differentiation factor that signals diverse phenotypes in normal and deregulated cellular growth and differentiation. See Milner, et al., (1989) Biochem. Biophys. Res. Commun. 165, 1096–1103; Rauvala, H. (1989) EMBO J. 8(10), 2933–2941; Li et al. (1990) Science 250, 1690–1694; Li et al., (1992) Biochem. Biophys. Res. Commun. 184: 427–432. PTN is nearly 50% identical with the retinoic acid-inducible factor midkine, which is also a growth and differentiation factor active in cultured fibroblasts, endothelial cells and epithelial cells. See Li et al., 1990, supra; Muramatsu et al.,(1993) Dev. Biol. 159, 392–402. Pleiotrophin gene expression is limited to specific cell types at different times during development; however, in adults, pleiotrophin gene expression is constitutive and limited to only a few cell populations except in sites of injury, when its expression is sharply increased. See Li et al. 1990, supra; Li et al., 1992 supra; Silos-Santiago et al, (1996) J. Neurobiol. 31, 283–296; Yeh et al., (1998) J. Neurosci. 18: 3699–3707.
The pleiotrophin (PTN) gene (Ptn) encodes an 18-kDa protein that is highly conserved among mammalian species and that functions as a weak mitogen and promotes neurite-outgrowth activity in vitro. Chauhan et al., Proc. Nat'l. Acad. Sci. 90: 679–682, 1993. PTN cDNA encodes a lysine-rich, highly basic protein of 168 amino acids with a 32-amino acid signal sequence that is highly conserved in bovine, rat, human, and chicken. Zhang et al. J. Biol. Chem. 272:16733–16736, 1997. The pleiotrophin gene is highly conserved among human, rat, bovine, and mouse species, and is developmentally regulated. Li et al., Biochem. Biophys. Res. Common. 184, 427–432, 1992. Li et al. (Science 250, 1690–1694, 1990) reported the isolation and sequence of the frill-length complementary DNA's (cDNA's) of the bovine, human, and rat genes of a heparin binding protein (i.e., pleiotrophin) with mitogenic activity toward rat and mouse fibroblasts. Comparison of predicted amino acid sequences of PTN from bovine, human, and rat revealed that PTN is conserved across the three species. Of 168 amino acid residues of PTN, 163 between bovine and human and 164 between rat and human are identical. The mature forms of bovine, human, and rat PTN exhibit overall 98 percent sequence similarity. Li et al., Science 250, 1690–1694, 1990. Zhang et al. describes the generation of a mouse PTN mutant gene construct containing sequences to encode mouse PTN residues −32 to +40 and a human wild type PTN expression vector containing a full-length human PTN cDNA fragment, and states that amino acid residues 1–40 (after cleavage of the signal peptide) of mouse and human PTN are identical, and thus the truncated PTN is equally effective in mouse and human lines. Zhang et al., J. Biol. Chem. 272: 16733–16736, 1997.
PTN also signals transformation; stable expression of an exogenous Ptn gene transforms NIH 3T3 cells and the Ptn-transformed NIH 3T3 cells form rapidly growing highly vascularized tumors in nude mice. Chauhan et al., (1993) PNAS USA 90: 679–682. Significantly, high level expression of the Ptn gene is found in many different human malignant tumors and in the cell lines that have been derived from these tumors; however, Ptn gene expression is not found in the normal cells from which the malignancy is derived. Fang, Hartmann et al. 1992, J. Biol. Chem. 267: 25889–97; Wellstein, Fang et al. 1992 J. Biol. Chem. 267: 2582–87; Tsutsui, Kadomatsu et al. 1993 Cancer Res. 53: 1281–85; Czubayko, Riegel et al. 1994, J. Biol. Chem. 269: 21358–63; Czubayko, Schulte et al. 1995, Breast Cancer Res Treat 36: 157–68; Czubayko, Schulte et al. 1996 PNAS USA 93: 14753–58; Brodeur, Nakagawara et al. 1997 J. Neurooncol. 31: 49–55; Zhang, Zhong et al. 1997 J. Biol. Chem. 272: 16733–36; Zhang and Deuel 1999 Curr Opin Hematol 6:44–50. Furthermore, high level expression of the Ptn gene may play a important role in developing a more aggressive phenotype in cancerous cells. Since it has also been shown that interruption of endogenous PTN signaling by a dominant negative PTN effector or a specific ribozyme reverses the malignant phenotype of human breast cancer cells (Zhang et al. 1997, J. Biol. Chem. 16733–36) and human melanoma cells (Czukayko et al., 1994 J. Biol. Chem. 269: 21358–63; Czubayko et al., 1996 PNAS USA 93: 14753–58), acquisition of PTN signaling during the course of these malignancies may trigger a more aggressive phenotype.
It is known that cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their immediate environment. These extracellular signals are important in the regulation of diverse cellular processes such as differentiation, contractility, secretion, cell division, cell migration, contact inhibition and metabolism. The extracellular molecules include, for example, hormones, growth factors or neurotransmitters, which may function as ligands that bind specific cell surface receptors. The binding of these ligands to their receptors triggers signal transduction, a cascade of reactions that brings about both the amplification of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above.
A central feature of signal transduction is the reversible phosphorylation of certain proteins. The phosphorylation or dephosphorylation of certain amino acid residues may trigger conformational changes in regulated proteins which results in the alteration of their biological properties. Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative activities, at that particular instant, of the protein linases and phosphatases that catalyze these reactions.
Protein kinases and phosphatases are classified according to the amino acid residues they act on, for example, the class of tyrosine kinases and phosphatases act on tyrosine residues. See Fischer, E. H. et al., (1991) Science 253: 401–406; Schlessinger, J. and Ullrich, A., (1992) Neuron 9:383–391; Ullrich, A. and Schlessinger, J., (1990) Cell 61:203–212. Protein kinases and phosphatases may further be defined as being receptors, i.e., the enzymes are an integral part of a transmembrane, ligand-binding molecule, or as non-receptors, meaning they respond to an extracellular molecule indirectly by being acted upon by a ligand-bound receptor.
The receptor class of protein tyrosine phosphatases (PTPs) is made up of high molecular weight, receptor-linked PTPases, termed RPTPases. Structurally resembling growth factor receptors, RPTPases consist of an extracellular, putative ligand-binding domain, a single transmembrane segment, and an intracellular catalytic domain (reviewed in Fischer et al., (1991) Science 253:401–406). Since the initial purification, sequencing and cloning of a protein tyrosine phosphatase (Thomas, M. L. et al., (1985) Cell 41:83), additional potential protein tyrosine phosphatases have been identified. One such example is a proteoglycan-type protein tyrosine phosphatase, named protein tyrosine phosphatase ζ/receptor-like PTP β (RPTP β/ζ). Recently, PTN was found to interact with the transmembrane RPTP β/ζ. See Maeda et al., (1996) J. Biol. Chem. 271: 21446–21452: Maeda, N. & Noda, M. (1998) J. Cell Biol. 142, 203–216; Milev et al., (1998) J. Biol. Chem. 273: 6998–7005.
The PTN gene is a protooncogene and is expressed in many human tumors such as breast cancer, neuroblastoma, glioblastoma, prostate cancer, lung cancer and Wilms' tumor and cell lines derived from human tumors. See Fang et al., (1992) J. Biol. Chem. 267: 25889–25897; Chauhan et al, (1993) Proc. Natl. Acad. Sci. USA 90: 679–682; Wellstein et al., (1992) J. Biol. Chem. 267: 2582–2587; Tsutsui et al., (1993) Cancer Res. 53:1281–1285; Nakagawara et al., (1995) Cancer Res. 55: 1792–1797. The importance of PTN in malignant cell growth was first established when introduction of the exogenous Ptn gene into NIH 3T3 cells and NRK cells led to morphological transformation, anchorage independent growth and tumor formation with significant neovascularization in vivo in the nude mouse. See Chauhan et al., 1993 PNAS USA 90: 679–82. It was subsequently shown that SW13 cells transformed by pleiotrophin also develop highly vascular tumors in the flanks of athymic nude mice. See Fang et al., 1992 J. Biol. Chem. 267: 258889–97. Further, interruption of PTN signaling has resulted in the reversal of the transformed phenotype of human breast cancer cells that constitutively express the PTN gene (Zhang et al., (1997) J. Biol. Chem. 272: 16733–16736) and effectively reverted the malignant phenotype of cultured human melanoma cells (Czubayko et al, (1994) J. Biol. Chem. 269: 21358–21363). It is believed that expression of the Ptn gene and its signaling pathway play a crucial regulatory role in many neoplasms of diverse origins. Thus, identification of the molecules and mechanisms of the PTN signaling pathway that are specific and crucial for tumor proliferation, angiogenesis and invasiveness would allow for the development of clinical applications and specific anti-tumor drugs to treat cancer. As such, a need presently exists for the identification of compounds or agents that disrupt or interfere with PTN signaling in order to influence malignant transformation and inhibit tumor growth and angiogenesis.
Further, PTN also induces neurite outgrowth from neurons (Rauvala 1989, supra; Li et al. 1990, supra) and glial process outgrowth from glial progenitor cells, suggesting that Ptn gene expression may influence a very broad range of functional activities. Since the pleiotrophin gene expression is upregulated by PDGF, PTN may act downstream of PDGF to mediate aspects of the PDGF signal. Thus, the activation of their respective signaling pathways is critical to the temporal maturation of oligodendrocyte progenitors and the properties of PTN suggest that PTN is ideally positioned to signal activation of genes important in maturation of glial elements at this critical time of development. As differentiation of oligodendrocytes is required for myelination of nerve fibers and consequently, important to nerve conduction, the determination of mechanisms for modulating PTN signaling during the differentiation of oligodendrocytes would be desirable. Accordingly, a need presently exists to determine the mechanism and molecules by which PTN signals in order to develop methods to treat and prevent nerve injury and demyelinating diseases.
While the molecules through which PTN signals have not to date been established, in addition to interacting with RPTP β/ζ, PTN has also been shown to bind to heparin, heparin sulfate proteoglycans and extracellular matrix. See Milner et al. 1989, supra; Rauvala, 1989 supra; Li et al., 1990, supra; Raulo et al., (1994) J. Biol. Chem. 269: 12999–13004; Maeda et al., (1996) J. Biol. Chem. 271: 21446–21452; Kinnunen et al., (1996) J. Biol. Chem. 271: 2243–2248. In addition to interacting with RPTP β/ζ, PTN induces tyrosine phosphorylation of a 190 kDa protein in PTN treated murine fibroblasts. See Li, Y. S. & Deuel, T. F. (1993) Biochem. Biophys. Res. Commun. 195: 1089–1095.
Thus, the interruption of PTN signaling impacts the events downstream in the signaling cascade such as cell proliferation and differentiation. Accordingly, there is presently a need to understand PTN signaling and the interaction between RPTP β/ζ and PTN in order to modulate the PTN signaling pathway to produce increased or decreased PTN activity in order to define compounds which useful in therapy and treating disease influenced by the expression of pleiotrophin such as cancer.